JP6639901B2 - New agricultural uses of Pseudomonas bacteria - Google Patents
New agricultural uses of Pseudomonas bacteria Download PDFInfo
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- JP6639901B2 JP6639901B2 JP2015252496A JP2015252496A JP6639901B2 JP 6639901 B2 JP6639901 B2 JP 6639901B2 JP 2015252496 A JP2015252496 A JP 2015252496A JP 2015252496 A JP2015252496 A JP 2015252496A JP 6639901 B2 JP6639901 B2 JP 6639901B2
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
Landscapes
- Catching Or Destruction (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Cultivation Of Plants (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Description
本発明はエンドファイトであるPseudomonas属細菌を植物に人為的に感染させて該植物に病虫害抵抗性を付与する方法、該植物の生育を促進させる方法、及び該植物の収量を増加させる方法に関する。 The present invention relates to a method for artificially infecting plants with Pseudomonas genus endophyte to impart pest and disease resistance to the plants, a method for promoting the growth of the plants, and a method for increasing the yield of the plants.
これまでの化学農薬を中心とした病害虫防除技術は、効率的な食糧確保に貢献してきた。ところが近年、栽培の効率性だけでなく、安心・安全といった領域を含めた無農薬、減農薬による環境保全型農業が望まれ、それに適合した病害虫防除技術（例えば微生物農薬）や生産性の向上技術が必要とされている。 Conventional pest control technologies, mainly chemical pesticides, have contributed to efficient food security. However, in recent years, not only the efficiency of cultivation but also the need for environment-friendly agriculture with no pesticides and reduced pesticides, including in areas such as safety and security, are required. Pest control technologies (for example, microbial pesticides) and productivity improvement technologies Is needed.
農業分野では、依然として、作物の生育促進には化学肥料が使用され、また、病害虫に対しては化学合成農薬が使用されている。また、一部では微生物や微生物が生産する物質を用いた微生物農薬も知られている。しかし、農薬の原体を化学合成により製造する際には、多量のエネルギーが投入されるという問題、環境への負荷が大きいという問題などがある。また、一方、現在使用されている環境負荷が小さい微生物農薬は高価であり、安定した防除効果が得られないため、品質や収量が減少してしまう問題がある。 In the agricultural field, chemical fertilizers are still used to promote the growth of crops, and chemically synthesized pesticides are used for pests. Microbial pesticides using microorganisms and substances produced by microorganisms are also known in part. However, when a raw material of an agricultural chemical is produced by chemical synthesis, there are a problem that a large amount of energy is input and a problem that a load on the environment is large. On the other hand, currently used microbial pesticides having a low environmental load are expensive and do not have a stable control effect, so that there is a problem that quality and yield are reduced.
このような状況において、植物に共生する微生物であるエンドファイトのなかに、病害虫の防除や収量の増加などの、植物に有益な特性を付与する微生物が報告されている（特許文献１〜９）。しかし、知られるエンドファイトの種類が少ないため、この分野での実用化のためには、実用化の可能性の高い微生物の探索と応用が求められている。 Under these circumstances, among endophytes, which are microorganisms symbiotic with plants, microorganisms that impart beneficial properties to plants, such as controlling pests and increasing yield, have been reported (Patent Documents 1 to 9). . However, since there are few known types of endfights, for practical use in this field, search and application of microorganisms having high possibility of practical use are required.
本発明は、微生物学的な手段によって農業上有用な植物に対し有益な性質を付与することを目的とする。 It is an object of the present invention to confer beneficial properties on agriculturally useful plants by microbiological means.
とりわけ、本発明に関わるPseudomonas属細菌をイネ科、マメ科、ユリ科などの植物に定着させて生育促進による増収技術、病虫害を抑制させる技術などは、これまで知られていない。 In particular, a technique of increasing the yield by promoting the growth of plants belonging to the genus Pseudomonas related to the present invention to plants such as grasses, legumes, and lilies and suppressing disease and pests has not been known.
本発明は、以下の特徴を包含する。
(1) シュードモナス（Pseudomonas）属に属し、農業上有用な植物体内に共生して該植物に病虫害抵抗性を付与する能力を有する細菌を、該植物に人為的に感染させる工程を含む、農業上有用な植物に病虫害抵抗性を付与する方法。
(2) シュードモナス（Pseudomonas）属に属し、農業上有用な植物体内に共生して該植物の収量を増加させる能力を有する細菌を、該植物に人為的に感染させる工程を含む、農業上有用な植物の収量を増加させる方法。
(3) シュードモナス（Pseudomonas）属に属し、農業上有用な植物体内に共生して該植物の生育を促進する能力を有する細菌を、該植物に人為的に感染させる工程を含む、農業上有用な植物の生育を促進する方法。
(4) 農業上有用な植物が、マメ科植物、イネ科植物、ユリ科植物、アブラナ科植物、キク科植物、ナス科植物、ウリ科植物及びセリ科植物から選択される植物である、(1)〜(3)のいずれかに記載の方法。
(5) 前記細菌が、配列番号1又は4に示す塩基配列と97%以上の同一性を有する塩基配列を含む16S rDNAを有する、(1)〜(4)のいずれか１項に記載の方法。
(6) 前記細菌がPseudomonas sp. MYK105（受託番号NITE P-02061）又はPseudomonas sp. MYK104（受託番号NITE P-02060）、或いは、農業上有用な植物体内に共生して該植物に病虫害抵抗性を付与する能力、該植物の収量を増加させる能力、及び／又は該植物の生育を促進する能力を有する、その変異株である、(1)〜(5)のいずれかに記載の方法。
(7) シュードモナス（Pseudomonas）属に属し、農業上有用な植物体内に共生して該植物に病虫害抵抗性を付与する能力、該植物の収量を増加させる能力、及び／又は該植物の生育を促進する能力を有する細菌を有効成分として含有する、農業上有用な植物用の微生物製剤。
(8) 農業上有用な植物が、マメ科植物、イネ科植物、ユリ科植物、アブラナ科植物、キク科植物、ナス科植物、ウリ科植物及びセリ科植物から選択される植物である、(7)に記載の微生物製剤。
(9) ユリ科植物が、タマネギである、(8)に記載の微生物製剤。
(10) 前記細菌が、配列番号1又は4に示す塩基配列と97%以上の同一性を有する塩基配列を含む16S rDNAを有する、(7)〜(9)のいずれかに記載の微生物製剤。
(11) 前記細菌がPseudomonas sp. MYK105（受託番号NITE P-02061）若しくはPseudomonas sp. MYK104（受託番号NITE P-02060）、又はその変異株である、(7)〜(10)のいずれかに記載の微生物製剤。
(12) Pseudomonas sp. MYK105（受託番号NITE P-02061）又はPseudomonas sp. MYK104（受託番号NITE P-02060）、或いは、農業上有用な植物体内に共生して該植物に病虫害抵抗性を付与する能力、該植物の収量を増加させる能力、及び／又は該植物の生育を促進する能力を有する、その変異株。
The present invention includes the following features.
(1) An agricultural method comprising the step of artificially infecting a plant with a bacterium belonging to the genus Pseudomonas and having the ability to coexist in an agriculturally useful plant and impart disease resistance to the plant. A method for imparting disease resistance to useful plants.
(2) an agriculturally useful bacterium comprising a step of artificially infecting the plant with a bacterium belonging to the genus Pseudomonas and having the ability to coexist in the agriculturally useful plant and increase the yield of the plant. How to increase plant yield.
(3) a step of artificially infecting the plant with a bacterium belonging to the genus Pseudomonas and having the ability to coexist in an agriculturally useful plant and to promote the growth of the plant; A method of promoting plant growth.
(4) Agriculturally useful plants are plants selected from legumes, gramineous plants, lilies, crucifers, asteraceae, solanaceae, cucurbits and apiaceae, The method according to any one of 1) to (3).
(5) The method according to any one of (1) to (4), wherein the bacterium has 16S rDNA containing a nucleotide sequence having 97% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 or 4. .
(6) The bacteria coexist in Pseudomonas sp. MYK105 (Accession number NITE P-02061) or Pseudomonas sp. The method according to any one of (1) to (5), which is a mutant thereof, which has the ability to impart C.I., the ability to increase the yield of the plant, and / or the ability to promote the growth of the plant.
(7) belongs to the genus Pseudomonas, and has the ability to coexist in an agriculturally useful plant to impart disease resistance to the plant, to increase the yield of the plant, and / or to promote the growth of the plant A microbial preparation for agriculturally useful plants, comprising a bacterium having the ability to do so as an active ingredient.
(8) An agriculturally useful plant is a plant selected from legumes, gramineous plants, lilies, cruciferous plants, asteraceous plants, solanaceous plants, cucurbitaceous plants and apiaceae plants, The microorganism preparation according to 7).
(9) The microbial preparation according to (8), wherein the lily plant is an onion.
(10) The microbial preparation according to any of (7) to (9), wherein the bacterium has 16S rDNA containing a nucleotide sequence having 97% or more identity to the nucleotide sequence shown in SEQ ID NO: 1 or 4.
(11) the bacterium is Pseudomonas sp. MYK105 (Accession number NITE P-02061) or Pseudomonas sp. MYK104 (Accession number NITE P-02060), or a mutant thereof, (7) to (10) The microbial preparation as described in the above.
(12) Pseudomonas sp. MYK105 (Accession number NITE P-02061) or Pseudomonas sp. MYK104 (Accession number NITE P-02060) A mutant thereof having the ability, the ability to increase the yield of the plant, and / or the ability to promote the growth of the plant.
本明細書において「シュードモナス（Pseudomonas）属細菌」は、農業上有用な植物体内に共生して植物に病虫害抵抗性を付与する能力、植物の収量を増加させる能力、及び／又は植物の生育を促進する能力を有する、エンドファイト細菌を指す。一般に、「エンドファイト」なる用語は、植物に共生する微生物をいう。 As used herein, the term "Pseudomonas bacterium" refers to an ability to coexist in an agriculturally useful plant to impart disease resistance to the plant, to increase the yield of the plant, and / or to promote the growth of the plant. Refers to endophytic bacteria that have the ability to Generally, the term "endophyte" refers to a microorganism that symbiotic with a plant.
本発明により、微生物学的な手段、すなわちエンドファイトとして使用可能なシュードモナス（Pseudomonas）属細菌（例えばNITE P-02061株又はNITE P-02060株）によって、以下のものに限定されないが、マメ科植物、イネ科植物、ユリ科植物、ナス科植物、ウリ科植物、アブラナ科植物、キク科植物及びセリ科植物から選択される植物について、該植物に病虫害抵抗性を付与すること、該植物の生育を促進すること、及び／又は該植物の収量を増加することが可能となる。例えばユリ科植物等に属する作物に対して、病虫害抵抗性を付与させるだけでなく、植物の生育を促進し、及び／又は植物の収量を増加させることが可能になる。後述の実施例で例証するように、本発明のシュードモナス（Pseudomonas）属細菌によって、タマネギ、キャベツ、コマツナ、レタス、及びナス等において、数%〜数十%の収量の増加が認められた。また、キャベツ軟腐病、キャベツ根こぶ病、キャベツのヨトウムシによる食害の被害率が約十%〜数十%低下した。 According to the present invention, depending on the microbiological means, ie Pseudomonas bacteria (eg NITE P-02061 or NITE P-02060 strain) which can be used as endophytes, legumes are not limited to: A plant selected from a gramineous plant, a lily plant, a solanaceous plant, a cucurbitaceous plant, a cruciferous plant, a chrysanthemum plant and an apiaceous plant, conferring the plant with pest and disease resistance, and growing the plant And / or increase the yield of the plant. For example, it is possible not only to impart pest and disease resistance to a crop belonging to a lily family or the like, but also to promote plant growth and / or increase plant yield. As exemplified in the examples described below, an increase in yield of several percent to several tens percent in onion, cabbage, komatsuna, lettuce, eggplant, and the like was observed by the bacteria of the genus Pseudomonas of the present invention. In addition, the damage rate of cabbage soft rot, cabbage root-knot disease, and cabbage was reduced by about 10% to several tens of%.
１. 細菌
本発明に用いることができる細菌は、シュードモナス（Pseudomonas）属に属する細菌、例えばPseudomonas koreensisであって、農業上有用な植物、例えば、マメ科植物、イネ科植物、ユリ科植物、アブラナ科植物、キク科植物、ナス科植物、ウリ科植物及びセリ科植物から選択される植物の体内に共生して、該植物に病虫害抵抗性を付与する能力、該植物の生育（生長）を促進する能力、及び／又は、該植物の収量を増加する能力を有する細菌であれば特に限定されない。
1. Bacteria A bacterium that can be used in the present invention is a bacterium belonging to the genus Pseudomonas, such as Pseudomonas koreensis, and is an agriculturally useful plant such as a leguminous plant, a gramineous plant, a lily family, and a rape. Ability to confer pest and disease resistance on plants, symbiotic in plants selected from the family plants, asteraceae plants, solanaceae plants, cucurbitaceous plants and apiaceae plants, and to promote the growth (growth) of the plants The bacterium is not particularly limited as long as it is a bacterium having the ability to do so and / or to increase the yield of the plant.
本明細書では、植物の収量は、例えば種子、葉（鱗茎を含む）、実、茎、根、花などの植物体構成成分の収量を指す。 In this specification, the yield of a plant refers to the yield of plant constituents such as seeds, leaves (including bulbs), fruits, stems, roots, and flowers.
本明細書では、シュードモナス（Pseudomonas）属細菌は、上記のいずれか１つ、２つ又は３つの能力を植物体に付与することができるエンドファイト細菌であり、自然界から単離された細菌だけでなく、そのような細菌に突然変異処理を施して産生された変異体も包含する。 As used herein, a Pseudomonas genus bacterium is an endophytic bacterium capable of conferring any one, two, or three of the above-mentioned abilities to a plant, and includes only a bacterium isolated from nature. And mutants produced by mutagenizing such bacteria.
上記細菌の具体例として、シュードモナス（Pseudomonas）属細菌である、Pseudomonas sp. MYK105（受託番号NITE P-02061；以下では、単に「NITE P-02061株」と称することもある。）又はPseudomonas sp. MYK104（受託番号NITE P-02060；以下では、単に「NITE P-02060株」と称することもある。）、或いは、上記のいずれか１つ又は複数の能力を有する、その変異株が挙げられる。 As a specific example of the bacterium, Pseudomonas sp. MYK105 (Accession No. NITE P-02061; hereinafter, it may be simply referred to as “NITE P-02061 strain”) or Pseudomonas sp. Which is a bacterium belonging to the genus Pseudomonas. MYK104 (accession number NITE P-02060; hereinafter, it may be simply referred to as “NITE P-02060 strain”), or a mutant thereof having any one or more of the above-mentioned capabilities.
上記のPseudomonas sp. MYK105は、自生しているユリ科植物から単離された菌株である。Pseudomonas sp. MYK105は、2015年5月29日を受託日として、ブダペスト条約下の国際寄託機関である、独立行政法人製品評価技術基盤機構、特許微生物寄託センター（〒292-0818千葉県木更津市かずさ鎌足2-5-8）に本出願人により寄託され、受託番号NITE P-02061が付与されている。 The above Pseudomonas sp. MYK105 is a strain isolated from a native Lily plant. Pseudomonas sp. MYK105 was established on May 29, 2015 under the Budapest Treaty as an international depositary organization, the National Institute of Technology and Evaluation, the Patented Microorganisms Depositary Center (Kazusa, Kisarazu, Chiba 292-0818, Japan) 2-5-8) and deposited with NITE P-02061.
このNITE P-02061株は、種々の細菌の属及び種について、16S rDNAの部分配列（本件の場合、Pseudomonas sp. MYK105（受託番号NITE P-02061）株の16S rDNAの対応する部分塩基配列（配列番号1）との比較）を用いて、相同性検索を行った結果、相同性の高い菌として、相同性が高いほうから順に、Pseudomonas sp. WXGSA1（Accession No.：KJ184866.1、相同性： 100％）、Pseudomonas koreensis strain JH18（Accession No.：KF424274.1、相同性： 99.9％）、Pseudomonas sp. WXGSY2（Accession No.：KJ184891.1、相同性： 99.9％）、Pseudomonas koreensis strain JH14（Accession No.：KF424272.1、相同性： 99.8％）、Pseudomonas koreensis strain RK18 (Accession No.：KC790278.1、相同性： 99.8％）が認められたことから、Pseudomonas属に属する細菌であることが判明した。また、この結果から、NITE P-02061株は、Pseudomonas koreensisである可能性が高いと考えられた。 This NITE P-02061 strain has a partial sequence of 16S rDNA (in this case, Pseudomonas sp. MYK105 (accession number NITE P-02061) corresponding to a partial base sequence of 16S rDNA for various genera and species of bacteria. As a result of a homology search using Pseudomonas sp. WXGSA1 (Accession No .: KJ184866.1, homology of : 100%), Pseudomonas koreensis strain JH18 (Accession No .: KF424274.1, homology: 99.9%), Pseudomonas sp. WXGSY2 (Accession No .: KJ184891.1, homology: 99.9%), Pseudomonas koreensis strain JH14 ( Accession No .: KF424272.1, homology: 99.8%), Pseudomonas koreensis strain RK18 (Accession No .: KC790278.1, homology: 99.8%), which indicates that the bacterium belongs to the genus Pseudomonas. found. From the results, NITE P-02061 strain was considered to be highly likely to be Pseudomonas koreensis.
また、NITE P-02061は、以下の基質資化性を有している。
また、上記のPseudomonas sp. MYK104は、圃場で栽培されたアブラナ科植物から単離された菌株である。Pseudomonas sp. MYK104は、2015年5月29日を受託日として、ブダペスト条約下の国際寄託機関である、独立行政法人製品評価技術基盤機構、特許微生物寄託センター（〒292-0818千葉県木更津市かずさ鎌足2-5-8）に本出願人により寄託され、受託番号NITE P-02060が付与されている。 The above Pseudomonas sp. MYK104 is a strain isolated from cruciferous plants cultivated in the field. Pseudomonas sp. MYK104 was established on May 29, 2015 under the Budapest Treaty as an international depositary organization, the National Institute of Technology and Evaluation, the Patent Microorganisms Depositary Center (Kazusa, Kisarazu, Chiba Prefecture, 292-0818, Japan). 2-5-8) and deposited with NITE P-02060.
このNITE P-02060株は、種々の細菌の属及び種について、16S rDNAの部分配列（本件の場合、Pseudomonas sp. MYK104（受託番号NITE P-02060）株の16S rDNAの対応する部分塩基配列（配列番号4）との比較）を用いて、相同性検索を行った結果、相同性の高い菌としてPseudomonas koreensis strain NHZ12 (Accession No.: KJ875595.1、相同性： 100%)、Pseudomonas koreensis strain SMs14 (Accession No.: JX485811.1、相同性： 100%)、Pseudomonas koreensis strain PSB24 (Accession No.: KJ875683.1、相同性： 100%)、Pseudomonas koreensis strain IARI-HHS2-32 (Accession No.: KF054782.1、相同性： 100%)、Pseudomonas sp. BF1-3 (Accession No.: KJ849233.1、相同性： 100%)が認められたことから、Pseudomonas属に属する細菌であることが判明した。また、この結果から、NITE P-02060株は、Pseudomonas koreensisである可能性が高いと考えられた。 This NITE P-02060 strain has a partial sequence of 16S rDNA (in this case, Pseudomonas sp. MYK104 (accession number NITE P-02060)) corresponding to a partial base sequence of 16S rDNA for various genera and species of bacteria. As a result of a homology search using Pseudomonas koreensis strain NHZ12 (Accession No .: KJ875595.1, homology: 100%), Pseudomonas koreensis strain SMs14 (Accession No .: JX485811.1, homology: 100%), Pseudomonas koreensis strain PSB24 (Accession No .: KJ875683.1, homology: 100%), Pseudomonas koreensis strain IARI-HHS2-32 (Accession No .: KF054782 .1, homology: 100%) and Pseudomonas sp. BF1-3 (Accession No .: KJ849233.1, homology: 100%) were confirmed to be bacteria belonging to the genus Pseudomonas. From the results, it was considered that NITE P-02060 strain was highly likely to be Pseudomonas koreensis.
また、NITE P-02060は、以下の基質資化性を有している。
なお、NITE P-02061株の16S rDNA（配列番号1からなる塩基配列）とNITE P-02060株の16S rDNA（配列番号4からなる塩基配列）は、99.9%の相同性を有する。 The 16S rDNA of NITE P-02061 strain (base sequence consisting of SEQ ID NO: 1) and the 16S rDNA of NITE P-02060 strain (base sequence consisting of SEQ ID NO: 4) have 99.9% homology.
本発明で使用できる細菌としては、Pseudomonas属細菌であるNITE P-02061株又はNITE P-02060株と同等の上記能力を有する細菌、例えば、（Pseudomonas koreensis等の）Pseudomonas属に属し、上記のいずれか１つ又は複数の能力を有し、及び、NITE P-02061株又はNITE P-02060株と同一又は同等の基質資化性等の性質を有する細菌や、Pseudomonas属に属し、上記のいずれか１つ又は複数の能力を有し、及び、配列番号1又は配列番号4に示す塩基配列又は該塩基配列と等価な配列を少なくとも一部分に含む16S rDNAを有する細菌が挙げられるがこれらには限定されない。ここで、同等の基質資化性を有するとは、NITE P-02061株又はNITE P-02060株の基質資化性と比べて、資化される基質及び／又は資化されない基質が、数個以下、例えば10個以下、9個以下、8個以下、7個以下、6個以下、5個以下、4個以下、3個以下、2個以下、又は1個異なることを意味する。また、配列番号1又は配列番号4に示す塩基配列と等価な配列とは、配列番号1又は4に示す塩基配列と95%以上、96%以上、好ましくは97%以上、98%以上、又は99%以上、さらに好ましくは99.5%以上、99.7%以上、99.8%以上、又は99.9%以上の同一性を有する塩基配列を意味する。同一性の値は、複数の塩基配列間の同一性を演算するソフトウェア（例えば、FASTA、DANASYS、及びBLAST）を用いてデフォルトの設定で算出した値を示す。 Examples of the bacterium that can be used in the present invention include bacteria having the above-mentioned ability equivalent to the strains of the genus Pseudomonas NITE P-02061 or NITE P-02060, for example, those belonging to the genus Pseudomonas (such as Pseudomonas koreensis) and any of the above. A bacterium having one or more abilities and having the same or equivalent substrate assimilation properties as the NITE P-02061 strain or the NITE P-02060 strain, or belonging to the genus Pseudomonas, A bacterium having one or more abilities and having a 16S rDNA containing at least a portion of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4 or a sequence equivalent to the nucleotide sequence, including but not limited to . Here, having the same substrate assimilation property means that compared to the substrate assimilation property of the NITE P-02061 strain or the NITE P-02060 strain, the number of assimilated substrates and / or unassimilated substrates is several. Hereinafter, for example, it means that there is a difference of 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 difference. In addition, the sequence equivalent to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4 is 95% or more of the nucleotide sequence shown in SEQ ID NO: 1 or 4, 96% or more, preferably 97% or more, 98% or more, or 99 % Or more, more preferably 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more nucleotide sequence. The value of identity indicates a value calculated by default using software for calculating identity between a plurality of base sequences (for example, FASTA, DANASYS, and BLAST).
さらにまた、NITE P-02061株又はNITE P-02060株が人為的に突然変異誘発処理されて産生されたその変異株であって、農業上有用な植物、例えばマメ科植物、イネ科植物、ユリ科植物、アブラナ科植物、キク科植物、ナス科植物、ウリ科植物及びセリ科植物から選択される植物、の体内に共生して該植物に病虫害抵抗性を付与する能力、該植物の生育を促進する能力、及び／又は該植物の収量を増加させる能力を有する変異株もまた、本発明で使用することができる。 Furthermore, the NITE P-02061 strain or the NITE P-02060 strain is a mutant thereof produced by artificial mutagenesis, and is an agriculturally useful plant such as a legume, a gramineous plant, and a lily. Family, cruciferous plants, asteraceae plants, solanaceous plants, cucurbitaceous plants, and plants selected from the plants of the family Umbelliferae, the ability of symbiosis in the body to impart disease resistance to the plants, the growth of the plants. Mutants that have the ability to promote and / or increase the yield of the plant can also be used in the present invention.
本発明で使用できるPseudomonas属細菌を自然界から分離するときには、農業上有用な植物の根、茎、葉などの植物体構成部から、該植物に共生する細菌類を培養により分離し、上記の能力のいずれかについて、並びに／或いは上記の配列番号1又は4に示す塩基配列との配列同一性等について、選抜試験を行い得る。 When the Pseudomonas genus bacterium that can be used in the present invention is isolated from the natural world, bacteria that are symbiotic with the plant are isolated by culturing from plant constituents such as roots, stems, and leaves of agriculturally useful plants, and the above-mentioned ability is determined. And / or for the sequence identity with the base sequence shown in SEQ ID NO: 1 or 4, and the like.
また、突然変異誘発処理を行う場合には、NITE P-02061株又はNITE P-02060株に対し任意の適当な変異原を用いて突然変異が行われ得る。 When performing the mutagenesis treatment, the NITE P-02061 strain or NITE P-02060 strain can be mutated using any appropriate mutagen.
ここで、「変異原」なる用語は、広義の意味を有し、例えば変異原作用を有する薬剤のみならずUV照射等の高エネルギー線照射のような変異原作用を有する処理も含むものとする。適当な変異原の例として、エチルメタンスルホネート、UV照射、ガンマ線照射、X線照射、重イオンビーム照射、N−メチル−N'−ニトロ−N−ニトロソグアニジン、ブロモウラシルのようなヌクレオチド塩基類似体、及びアクリジン類が挙げられるが、他の任意の効果的な変異原もまた使用され得る。 Here, the term “mutagen” has a broad meaning and includes not only a drug having a mutagenic effect but also a treatment having a mutagenic effect such as irradiation with high energy rays such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, gamma irradiation, X-ray irradiation, heavy ion beam irradiation, nucleotide base analogs such as N-methyl-N'-nitro-N-nitrosoguanidine, bromouracil , And acridines, but any other effective mutagen may also be used.
或いは、細菌に変異を導入する他の手段には、遺伝子組換え法を利用する方法がある。特に、虫害対策の場合、細菌ゲノムへの、忌避物質の産生を可能とする遺伝子若しくはcDNAの導入、Btトキシンなどの殺虫蛋白質をコードする遺伝子若しくはcDNAの導入などが挙げられる。 Alternatively, another means for introducing a mutation into a bacterium is to use a gene recombination method. In particular, in the case of insect pest control, introduction of a gene or cDNA capable of producing a repellent substance into a bacterial genome, introduction of a gene or cDNA encoding an insecticidal protein such as Bt toxin, and the like can be mentioned.
本発明に用いられる細菌は、振とう培養等の通常の培養法により、Pseudomonas属細菌について通常使用されるような条件下で培養されうる。培養に用いる培地としては炭素源としてグルコース、シュークロース、デンプン、デキストリンなどの糖類を、窒素源として硫酸アンモニウム、塩化アンモニウム、硝酸アンモニウム等のアンモニウム塩、硝酸塩等の無機窒素源、又は、酵母エキス、コーン・スティープ・リーカー、肉エキス、小麦胚芽、ポリペプトン、サトウキビ絞り粕（バカス）、ビールカス、大豆粉、米糠、魚粉等の有機窒素源を、無機塩としてリン酸一カリ、硫酸マグネシウム、硫酸マンガン、硫酸第一鉄等の、リン、カリウム、マンガン、マグネシウム、鉄等を含む塩類を、それぞれ含有する合成又は天然の培地が挙げられる。培養温度は、通常、20〜37℃、好ましくは27〜32℃で、12〜48時間、好気的条件下で行うことができる。 The bacterium used in the present invention can be cultured by a usual culturing method such as shaking cultivation under conditions generally used for Pseudomonas bacteria. As the medium used for the culture, saccharides such as glucose, sucrose, starch, and dextrin are used as a carbon source; ammonium sulfates such as ammonium sulfate, ammonium chloride, and ammonium nitrate; inorganic nitrogen sources such as nitrate; or yeast extract, corn Organic nitrogen sources such as steep leaker, meat extract, wheat germ, polypeptone, sugarcane pomace, beer cass, soybean flour, rice bran, fish meal, etc. Synthetic or natural culture media each containing salts containing phosphorus, potassium, manganese, magnesium, iron and the like, such as monoiron. The cultivation temperature is usually 20 to 37 ° C, preferably 27 to 32 ° C, for 12 to 48 hours under aerobic conditions.
本発明の方法には、細菌の培養液をそのまま使用することができるが、細菌の培養液を膜分離、遠心分離、濾過分離等の方法により分離した、細菌の高濃度物を用いることもできる。 In the method of the present invention, a bacterial culture solution can be used as it is, but a bacterial culture solution separated by a method such as membrane separation, centrifugation, or filtration can be used, and a high concentration of bacteria can also be used. .
本発明の方法ではまた、細菌の培養液を乾燥させたものを使用することができる。また、細菌の培養液を活性炭、珪藻土、タルク、ゼオライト、ピートモス、パーライト、ベントナイト、モンモリナイト、バーミュキュライト等の多孔吸着体に吸着させ乾燥させたものを使用することができる。多孔吸着体は、１種類でもよいし、複数の担体を組合せて用いてもよい。乾燥方法は通常の方法でよく、例えば凍結乾燥又は減圧乾燥でよい。これらの乾燥物は乾燥後さらにボールミル等の粉砕手段で粉砕されてもよい。 In the method of the present invention, a dried bacterial culture can also be used. Further, a bacterial culture solution that has been adsorbed on a porous adsorbent such as activated carbon, diatomaceous earth, talc, zeolite, peat moss, perlite, bentonite, montmorillonite, and vermiculite and dried can be used. The porous adsorbent may be of one type or a combination of a plurality of carriers. The drying method may be an ordinary method, for example, freeze drying or drying under reduced pressure. These dried products may be further pulverized by a pulverizing means such as a ball mill after drying.
細菌は、上記の培養液、高濃度物又は乾燥物としてそれ自体単独で本発明の用途に用いることができるが、更なる他の任意成分と組み合わせて通常の微生物製剤と同様の形態（例えば粉剤、水和剤、粒剤、乳剤、液剤、懸濁液、フロアブル剤、塗布剤等の形態）に製剤化して用いることもできる。組み合わせて使用することができる任意成分としては例えば固体担体、補助剤のような植物への適用が許容される材料が挙げられる。 The bacterium can be used for the purpose of the present invention by itself as the above-mentioned culture solution, high-concentration product or dried product, but it can be used in combination with other optional components in the same form as ordinary microbial preparations (for example, powders). , Wettable powders, granules, emulsions, liquids, suspensions, flowables, coatings and the like). Optional components that can be used in combination include, for example, materials that are acceptable for application to plants, such as solid carriers and adjuvants.
２．農業上有用な植物
本発明の方法で使用可能な対象植物は、以下のものに限定されないが、例えばマメ科植物、イネ科植物、ユリ科植物、アブラナ科植物、キク科植物、ナス科植物、ウリ科植物及びセリ科植物から選択される農業上有用な植物が挙げられる。
2. Agriculturally useful plants Target plants that can be used in the method of the present invention include, but are not limited to, legumes, grasses, lilies, crucifers, asteraceae, solanaceae, Agriculturally useful plants selected from Cucurbitaceae and Umbelliferae.
本明細書で使用される「農業上有用な植物」は、例えば野菜類、穀類植物などの作物、果樹などの農業において生産対象となる植物を指す。 As used herein, “agriculturally useful plants” refers to plants that are produced in agriculture, such as crops such as vegetables and cereal plants, and fruit trees.
イネ科植物としては、例えばイネ、コムギ、オオムギ、ライムギ、ライコムギ、ハトムギ、ソルガム、エンバク、トウモロコシ、サトウキビ、アワ、ヒエなどの穀類が挙げられる。イネ科植物としてはさらに、例えばシバ、バッファローグラス、バミューダグラス、ウィーピンググラス、センチピードグラス、カーペットグラス、ダリスグラス、キクユグラス、セントオーガスチングラスなどの飼料又は牧草が挙げられる。 Gramineae include, for example, cereals such as rice, wheat, barley, rye, triticale, barley, sorghum, oat, corn, sugarcane, millet, and millet. Examples of grasses further include feed or grass such as grass, buffalo grass, bermudagrass, weeping grass, centipede grass, carpet grass, darisgrass, kikuyugrass, and St. Augustinegrass.
マメ科植物としては、例えばダイズ、アズキ、ラッカセイ、インゲンマメ、エンドウマメ、ハナマメ、ソラマメ、ササゲ、ヒヨコマメ、リョクトウ、レンズマメ、ライマメ、バンバラマメが挙げられる。 Examples of leguminous plants include soybean, adzuki bean, peanut, kidney bean, pea, lentil, broad bean, cowpea, chickpea, mung bean, lentil, lime bean, and bambara bean.
ユリ科植物としては、例えばタマネギ、ネギ、ラッキョウ、ニンニク、ニラ、アサツキ、ユリ、アスパラガス、エシャロット、ワケギなどが挙げられる。 Examples of the lily family include onion, leek, rakkyo, garlic, leek, asatsuki, lily, asparagus, shallot, scallion and the like.
アブラナ科植物としては、例えばアブラナ、カブ、チンゲンサイ、ノザワナ、カラシナ、タカナ、コブタカナ、水菜、コールラビー、ルッコラ、クレソン、タアサイ、カリフラワー、キャベツ、ケール、ハクサイ、コマツナ、ダイコン、ハツカダイコン、ブロッコリー、メキャベツ、ワサビ、セイヨウワサビ、シロイヌナズナが挙げられる。 Examples of the Brassicaceae plants include, for example, oilseed rape, turnip, bok choy, nozawana, mustard, takana, kobutana, mizuna, kohlrabi, arugula, watercress, taasai, cauliflower, cabbage, kale, Chinese cabbage, komatsuna, radish, radish, broccoli, mekabetsu, wasabi. , Horseradish and Arabidopsis.
キク科植物としては、例えばレタス、サニーレタス、シュンギク、キクなどが挙げられる。 Examples of the Asteraceae plants include lettuce, sunny lettuce, chrysanthemum, and chrysanthemum.
ナス科植物としては、例えばナス、トマト、ピーマン、シシトウ、トウガラシ、ジャガイモ、クコ、パプリカ、ハラペーニョ、ハバネロなどが挙げられる。 Examples of solanaceous plants include eggplant, tomato, bell pepper, shishito, capsicum, potato, wolfberry, paprika, jalapeno, habanero and the like.
ウリ科植物としては、例えばキュウリ、カボチャ、ゴーヤ、スイカ、ズッキーニ、トウガン、ヘチマ、メロン、ユウガオなどが挙げられる。 Examples of cucurbits include cucumber, pumpkin, bitter gourd, watermelon, zucchini, castor, loofah, melon, yugao and the like.
セリ科植物としては、ニンジン、ミツバ、パセリ、セロリ、セリ、アシタバ、スープセロリ、チャーベル、フェンネルなどが挙げられる。 Examples of the Apiaceae plant include carrot, honeybee, parsley, celery, celery, ashitaba, soup celery, charbell, fennel and the like.
３．病害虫
本発明の細菌が、上記の農業上有用な植物の体内に共生することによって病虫害抵抗性を付与する、対象の植物病及び害虫として、以下のものに限定されないが、例えば以下のものが挙げられる。
3. Diseases and pests The bacterium of the present invention imparts pest and disease resistance by coexisting in the body of the above-mentioned agriculturally useful plant.Plant diseases and pests of interest are not limited to the following, but include, for example, the following: Can be
植物病の例は、以下のとおりである。
イネ科植物の植物病として、例えば幼鞘褐変病(Sheath brown rot of rice)、いもち病、白葉枯病、苗立枯病、紋枯病、ばか苗病などが挙げられる。
Examples of plant diseases are as follows.
Examples of plant diseases of grasses include grass rot of rice (Sheath brown rot of rice), blast disease, white blight, seedling blight, sheath blight, falcon seedling blight, and the like.
マメ科植物の植物病として、例えば灰色かび病、さび病、うどんこ病などが挙げられる。 Examples of legume plant diseases include gray mold, rust, and powdery mildew.
ユリ科植物の植物病として、例えば軟腐病、べと病、い縮病、乾腐病、さび病、いちょう病、茎枯れ病、はん点病などが挙げられる。 Plant diseases of lily plants include, for example, soft rot, downy mildew, wilt, dry rot, rust, biloba, stem wilt, and spot disease.
アブラナ科の植物病として、例えば軟腐病、黒はん細菌病、苗立枯れ病、べと病、い黄病、モザイク病、根こぶ病、白はん病、しり腐れ病などが挙げられる。 Examples of plant diseases of the Brassicaceae include soft rot, black rot bacterial disease, seedling blight, downy mildew, yellow scab, mosaic disease, clubroot, white rot, and rot.
キク科植物の植物病として、例えばモザイク病、軟腐病、腐敗病、うどんこ病、べと病などが挙げられる。 Examples of plant diseases of Asteraceae include mosaic disease, soft rot, rot, powdery mildew, downy mildew, and the like.
ナス科植物の植物病として、例えばモザイク病、黄化えそ病（TSWV）、青枯れ病、かいよう病、褐色根腐れ病、苗立枯れ病、うどんこ病、半身いちょう病、葉かび病などが挙げられる。 Plant diseases of solanaceous plants include, for example, mosaic disease, yellow wilt (TSWV), bacterial wilt, mildew, brown root rot, seedling wilt, powdery mildew, half body wilt, leaf mold, etc. Is mentioned.
ウリ科植物の植物病として、例えば青枯れ病、苗立枯れ病、うどんこ病、べと病、つる割病などが挙げられる。 Plant diseases of Cucurbitaceae include, for example, bacterial wilt, seedling wilt, powdery mildew, downy mildew, and vine disease.
セリ科植物の植物病として、例えばモザイク病、軟腐病、黒葉枯れ病、はん点病、うどんこ病、菌核病などが挙げられる。 Examples of plant diseases of the Umbelliferae plant include mosaic disease, soft rot, black leaf blight, spot disease, powdery mildew, and sclerotium disease.
害虫の例は、以下のとおりである。虫害は、摂食、吸汁、ウイルス媒介などである。
イネ科植物の害虫として、例えばドロオイムシ、カメムシ、ニカメイガ、イチモンジセセリ、コブノメイガ、イネヨトウ、アワヨトウ、スジキリヨトウ、フタオビコヤガ、イネクビボソハムシ、その他ハムシ類、イネカラバエ、イネハモグリバエ、イネヒメハモグリバエ、コウモリガ、ミノガ、イネシンガレセンチュウ、その他センチュウ類、イネミズゾウムシ、コメツキムシ類、コガネムシ類、バッタ類、スクミリンゴガイ、シロトビムシ類、ガガンボ類、タマバエ類、セジロウンカ、トビイロウンカ、ヒメトビウンカ、その他ウンカ類、ヨコバイ類、フキムシ類、アブラムシ類、アザミウマ類などが挙げられる。
Examples of pests are as follows. Insect damage is feeding, sucking, virus-borne and the like.
Examples of pests of Poaceae plants include, for example, beetles, stink bugs, bark beetles, rice squirrels, potato beetles, rice armyworms, locust beetles, sycamore, other flea beetles, rice flies, rice moss, rice moss Gall nematode, other nematodes, rice water weevil, click beetles, scarab beetles, grasshoppers, scallops, white beetles, crane flies, tamaflies, white-spotted planthoppers, brown planthoppers, brown planthoppers, other planthoppers, flies and insects And the like.
マメ科の害虫として、例えばホソヘリカメムシ、マメコガネ、ウコンノメイガ、アズキノメイガ、ヒメコガネ、マメノメイガ、マメドクガ、アオアツバ、イチジクキンウワバ、フタスジヒメハムシ、ブチヒゲカメムシ、ツマジロカメムシ、ヨモギエダシャク、ウリハムシモドキ、ナシケンモン、ミツモンキンウワバ、ヒメシロモンドクガ、モンキチョウ、ナカグロカスミカメ、ホシハラビロヘリカメムシ、ベッコウハゴロモなどが挙げられる。 As pests of the legume family, for example, Helicoverpa, beetles, turmeric beetle, azukimemeiga, himekogane, bememeomea, beetle moth, aotsuba, figkinkiniwaba, futasujimehimeshi beetle, beete beetle, tsumejimomidomi, beetle beetle Nishikenmon, Mitsumonkiniwaba, Himeshiromondouga, Monkicho, Nakaguro Kasumi turtle, Hoshiharabirohelikamushi, Bekkouhagoromo and the like.
ユリ科植物の害虫として、例えばネギアザミウマ、ネギハモグリバエ、アザミウマ類、ヨトウムシなどが挙げられる。 Examples of pests of the lily family include thrips, leeks, thrips, and armyworms.
アブラナ科の害虫として、例えばコナガ、モンシロチョウ、オオモンシロチョウ、ハイマダラノメイガ、カブラヤガ、タマナヤガ、ヨトウムシ類、ハモグリバエ類、カブラハバチ類、キスジノミハムシ、ヤサイゾウムシ、アブラムシ類、アザミウマ類などが挙げられる。 Pests of the Brassicaceae family include, for example, diamondback moths, cabbage white butterflies, pieris brassicae, bombyx scrophulari, mosquito moths, tamananaaga, armyworms, scrophulariid flies, wasps, wasp flea beetles, beetles, aphids and thrips.
キク科植物の害虫として、例えばシロシタヨトウ、イチジクキンウワバ、タマナギンウワバ、ホソバセダカモクメ、ナシケンモン、ヨモギエダシャク、オオトビスジエダシャク、ナガメなどが挙げられる。 Examples of the pests of the Asteraceae plants include white armyworm, fig king walva, tamaganin wava, hososubedakamokume, nashikenmon, mugwort, and otobisujiedashaku, Nagame.
ナス科植物の害虫として、例えばオオタバコガ、オオニジュウヤホシテントウ、ナスノミハムシ、アズキノメイガ、イチジクキンウワバ、トホシテントウなどが挙げられる。 Examples of the insect pests of the Solanaceae plant include Citrus pallidum, A. pallidum, Echinacea purpurea, Azukimeiga, Fig.
ウリ科植物の害虫として、例えばオオタバコガ、ウリキンウワバ、ウリハムシ、アブラムシ類などが挙げられる。 Pests of Cucurbitaceae include, for example, Helicoverpa armigera, Urikin'waba, Urihamushi, Aphids and the like.
セリ科植物の害虫として、例えばウリハムシモドキ、キアゲハ、ミツモンキンウワバなどが挙げられる。
その他、カタツムリ、センチュウなどが挙げられる。
Examples of pests of the Umbelliferae plant include, for example, Urihamushimodoki, Papilio machaon, and Mitsumonkinwaba.
Other examples include snails and nematodes.
４．病虫害抵抗性の付与、収量増加、及び生育促進のための微生物学的方法
本発明の方法は、Pseudomonas属に属し、農業上有用な上記の植物の体内に共生して該植物に病虫害抵抗性を付与する能力、該植物の収量を増加する能力、及び／又は、該植物の生育を促進する能力を有する細菌を、該植物に人為的に感染させる工程を含む。
4. The method of the present invention belongs to the genus Pseudomonas and coexists in the above-mentioned agriculturally useful plants to impart pest and insect resistance to the plants. Artificially infecting the plant with a bacterium having the ability to impart, increase the yield of the plant, and / or promote the growth of the plant.
植物に病虫害抵抗性を付与することは、植物が病害虫による影響、例えば摂食、吸汁、病原菌病、ウイルス病などによる植物被害、を抑制又は防止することに導く。理論により拘束されることを望むものではないが、本発明に係る細菌は病原体に直接作用するのではなく、植物自身が有する防御機能の活性化によって、植物に病虫害抵抗性を付与すると考えられる。抵抗性誘導により生じる反応として、限定されるものではないが、過敏感反応、抗菌タンパク質及び抗害虫性タンパク質の生産、パピラの形成、並びに細胞壁の硬化等が挙げられる。一般に、植物の抵抗性が誘導されれば、広範な病虫害に対して抵抗性を示すことが知られている。 Providing a plant with pest resistance leads to a plant that suppresses or prevents the effects of the pest, for example, plant damage due to feeding, sucking, pathogenic fungal disease, viral disease and the like. While not wishing to be bound by theory, it is believed that the bacteria of the present invention do not act directly on pathogens, but rather confer pest and disease resistance on plants by activating the defense functions of the plants themselves. Reactions resulting from resistance induction include, but are not limited to, hypersensitivity reactions, production of antibacterial and anti-pest proteins, formation of papillae, hardening of cell walls, and the like. In general, it is known that if resistance of a plant is induced, it will exhibit resistance to a wide range of pests and diseases.
植物の収量を増加することは、例えば、野菜であれば、例えば葉（鱗茎を含む）、根、根茎、種子、花などの食用部分を、穀類であれば、種子を、果樹であれば、実を、それぞれ増収穫させることを意味する。 To increase the yield of plants, for example, if vegetables, leaves (including bulbs), roots, rhizomes, seeds, edible parts such as flowers, cereals, seeds, fruit trees, It means increasing the yield of each fruit.
植物の生育（生長）を促進することは、本発明の細菌を接種しない対照と比較して植物の成長を早めることを意味する。 Promoting plant growth (growth) means accelerating plant growth compared to a control not inoculated with the bacteria of the present invention.
植物への施用方法としては、種子コート、幼植物への潅注、塗布、植物根部の菌液への浸漬、又は噴霧処理する方法などが挙げられる。特に、種子又は植物体に人為的に傷を付け菌液の噴霧処理、塗布する方法が好ましい。その他の施用条件としては播種時、育苗期など圃場定植前に施用することが望ましい。また、さらに圃場栽培中に植物、場合により植物根部周囲の土壌、に噴霧処理することで効果の高発現が期待できる。 Examples of the method of application to plants include a method of seed coating, irrigation and application to young plants, immersion of plant roots in a fungal solution, or spraying. In particular, a method of artificially damaging a seed or a plant body and spraying and applying a bacterial solution is preferable. As other application conditions, it is desirable to apply before planting in a field, such as at the time of sowing or raising a seedling. Further, high effects can be expected by spraying the plant, and in some cases, the soil around the root of the plant during field cultivation.
1つの例として、本発明の細菌の、植物への人為的な感染は、圃場に植えつける前の幼苗（例えば１〜4枚の本葉が出た時期の苗）に菌液を散布することにより行うことができる。散布後約3〜15日目に、苗を圃場に定植し得る。植物体内に侵入した菌がやがて、植物に共生するようになると考えられる。 As one example, artificially infecting a plant with the bacterium of the present invention is to spray a bacterial solution on a seedling (for example, a seedling having 1 to 4 true leaves) before planting in a field. Can be performed. About 3 to 15 days after application, the seedlings can be planted in the field. It is thought that the bacteria that have entered the plant eventually become symbiotic with the plant.
菌液の濃度は、１×105〜1×109個/ml又はそれ以上であってよいが、これらの濃度範囲に限定されない。菌を懸濁する媒体は、水又は培地であることが好ましい。通常、高濃度菌液を水又は培地で所定濃度に希釈して使用することができる。 The concentration of the bacterial solution may be 1 × 10 5 to 1 × 10 9 cells / ml or more, but is not limited to these concentration ranges. The medium in which the bacteria are suspended is preferably water or a medium. Usually, a highly concentrated bacterial solution can be used after being diluted to a predetermined concentration with water or a medium.
本発明の方法は、具体的には、次の第1から第3の態様からなる。
本発明の方法は、第1の態様により、シュードモナス（Pseudomonas）属に属し、農業上有用な植物体内に共生して該植物に病虫害抵抗性を付与する能力を有する細菌を、該植物に人為的に感染させる工程を含む、農業上有用な植物に病虫害抵抗性を付与する方法を提供する。
The method of the present invention specifically includes the following first to third aspects.
According to the first aspect, the method of the present invention is characterized in that a bacterium belonging to the genus Pseudomonas, which has the ability to coexist in an agriculturally useful plant and impart pest and disease resistance to the plant, is artificially added to the plant. A method for imparting pest and disease resistance to an agriculturally useful plant, comprising the step of infecting the plant with a disease.
本発明の方法は、第2の態様により、シュードモナス（Pseudomonas）属に属し、農業上有用な植物体内に共生して該植物の収量を増加させる能力を有する細菌を、該植物に人為的に感染させる工程を含む、農業上有用な植物の収量を増加させる方法を提供する。 According to the second aspect, the method of the present invention is to artificially infect a plant with a bacterium belonging to the genus Pseudomonas and having the ability to coexist in an agriculturally useful plant and increase the yield of the plant. Providing a method for increasing the yield of an agriculturally useful plant, comprising the step of:
本発明の方法は、第3の態様により、シュードモナス（Pseudomonas）属に属し、農業上有用な植物体内に共生して該植物の生育を促進する能力を有する細菌を、該植物に人為的に感染させる工程を含む、農業上有用な植物の生育を促進する方法を提供する。 According to the third aspect, the method of the present invention is to artificially infect a plant with a bacterium belonging to the genus Pseudomonas and having the ability to coexist in an agriculturally useful plant and promote the growth of the plant. And a method for promoting the growth of an agriculturally useful plant.
５．微生物製剤
本発明はさらに、シュードモナス（Pseudomonas）属に属し、農業上有用な植物体内に共生して該植物に病虫害抵抗性を付与する能力、該植物の収量を増加させる能力、及び／又は、該植物の生育を促進する能力を有する細菌を有効成分として含有する、農業上有用な植物用の微生物製剤を提供する。
5. Microbial preparations The present invention further belongs to the genus Pseudomonas, which is capable of symbiotic in an agriculturally useful plant to impart disease resistance to the plant, increase the yield of the plant, and / or Provided is an agriculturally useful microbial preparation for a plant, comprising a bacterium capable of promoting plant growth as an active ingredient.
農業上有用な植物の例は、上で具体的に例示した、マメ科植物、イネ科植物、ユリ科植物、ナス科植物、ウリ科植物、アブラナ科植物、キク科植物及びセリ科植物から選択される植物である。 Examples of agriculturally useful plants are selected from the legumes, gramineous plants, lilies, solanaceous plants, cucurbitaceous plants, cruciferous plants, asteraceae plants and agaric plants specifically exemplified above. Plants.
好ましい細菌は、Pseudomonas sp. MYK105（受託番号NITE P-02061）若しくはPseudomonas sp. MYK104（受託番号NITE P-02060）、又はその変異株である。 A preferred bacterium is Pseudomonas sp. MYK105 (Accession No. NITE P-02061) or Pseudomonas sp. MYK104 (Accession No. NITE P-02060), or a mutant thereof.
微生物製剤は、細菌の高濃度物、細菌の培養液を乾燥させたものなどを使用することができる。また、細菌の培養液を活性炭、珪藻土、タルク、ゼオライト、ピートモス、パーライト、ベントナイト、モンモリナイト、バーミュキュライト等の多孔吸着体に吸着させ乾燥させたものを使用することができる。多孔吸着体は、１種類でもよいし、複数の担体を組合せて用いてもよい。乾燥方法は通常の方法でよく、例えば凍結乾燥、減圧乾燥でよい。これらの乾燥物は乾燥後さらにボールミル等の粉砕手段で粉砕されてもよい。 As the microbial preparation, a high concentration of bacteria, a dried culture of bacteria, and the like can be used. Further, a bacterial culture solution that has been adsorbed on a porous adsorbent such as activated carbon, diatomaceous earth, talc, zeolite, peat moss, perlite, bentonite, montmorillonite, and vermiculite and dried can be used. The porous adsorbent may be of one type or a combination of a plurality of carriers. The drying method may be a usual method, for example, freeze-drying or vacuum drying. These dried products may be further pulverized by a pulverizing means such as a ball mill after drying.
細菌は、上記の培養液、高濃度物又は乾燥物としてそれ自体単独で本発明の用途に用いることができるが、更なる他の任意成分と組み合わせて通常の微生物製剤と同様の形態（例えば粉剤、水和剤、粒剤、乳剤、液剤、懸濁液、フロアブル剤、塗布剤等の形態）に製剤化して用いることもできる。組み合わせて使用することができる任意成分としては例えば固体担体、補助剤のような植物への適用が許容される材料が挙げられる。 The bacterium can be used for the purpose of the present invention by itself as the above-mentioned culture solution, high-concentration product or dried product, but it can be used in combination with other optional components in the same form as ordinary microbial preparations (for example, powders). , Wettable powders, granules, emulsions, liquids, suspensions, flowables, coatings and the like). Optional components that can be used in combination include, for example, materials that are acceptable for application to plants, such as solid carriers and adjuvants.
本発明はさらに、Pseudomonas sp. MYK105（受託番号NITE P-02061）又はPseudomonas sp. MYK104（受託番号NITE P-02060）、或いは、農業上有用な植物体内に共生して該植物に病虫害抵抗性を付与する能力、該植物の収量を増加させる能力、及び／又は、該植物の生育を促進する能力を有する、その変異株を提供する。 The present invention further provides Pseudomonas sp. MYK105 (Accession No. NITE P-02061) or Pseudomonas sp. MYK104 (Accession No. NITE P-02060), or coexist in an agriculturally useful plant to make the plant resistant to pests and insects. Provided is a mutant thereof having the ability to impart, the ability to increase the yield of the plant, and / or the ability to promote the growth of the plant.
本発明の細菌は、振とう培養等の通常の培養法により、Pseudomonas属細菌について通常使用されるような、上記の条件下で培養されうる。 The bacterium of the present invention can be cultured by a conventional culturing method such as shaking culturing under the above-mentioned conditions, such as those usually used for Pseudomonas bacteria.
以下に、実施例を挙げて本発明をさらに具体的に説明するが、本発明の技術的範囲は、それらの実施例によって制限されないものとする。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by the examples.
［実施例１］
＜Pseudomonas sp. MYK105（受託番号NITE P-02061）株、及びPseudomonas sp. MYK104（NITE P-02060）株の単離＞
自生しているユリ科植物を採取し、切断した。切断したユリ科植物を70%エタノールに30秒、2.5%次亜塩素酸ナトリウム溶液に5分浸すことにより表面殺菌を行った。その後、植物体を乳鉢に移し滅菌した生理食塩水1mlと海砂を適量加えながら磨砕した。磨砕した上澄みを100μl、NA(Nutrient Agar)培地に塗布し、30℃、数日間培養後、シングルコロニーを単離することにより標題のNITE P-02061株を得た。
[Example 1]
<Isolation of Pseudomonas sp. MYK105 (Accession No. NITE P-02061) strain and Pseudomonas sp. MYK104 (NITE P-02060) strain>
Indigenous lilies were collected and cut. Surface disinfection was performed by immersing the cut lily plants in 70% ethanol for 30 seconds and 2.5% sodium hypochlorite solution for 5 minutes. Thereafter, the plants were transferred to a mortar and ground with 1 ml of sterile physiological saline and an appropriate amount of sea sand. 100 μl of the crushed supernatant was applied to NA (Nutrient Agar) medium, cultured at 30 ° C. for several days, and a single colony was isolated to obtain the title NITE P-02061 strain.
圃場で栽培されたキャベツを採取し、その茎を切断した。表面殺菌、磨砕、シングルコロニーの単離を上記と同様に行い、標題のNITE P-02060株を得た。 Cabbage cultivated in the field was collected and its stem was cut. Surface sterilization, trituration, and isolation of a single colony were performed in the same manner as described above to obtain the title NITE P-02060 strain.
［実施例２］
＜Pseudomonas sp. MYK105（受託番号NITE P-02061）株、及びPseudomonas sp. MYK104（NITE P-02060）株の同定＞
１）方法
Pseudomonas sp. MYK105株菌体を0.85%NaCl水溶液500μlに懸濁し、遠心後に上清を除去した。菌体に滅菌水20μl、BL buffer（40ｍM Tris、1%Tween20、0.5% Nonidet P-40、1mM EDTA、pH 8.0）25μl、Proteinase K（1mg/ml）5μlを加え、軽く懸濁した。60℃、20分間放置後に105℃で5分間放置した。遠心し、上清をDNA抽出液とした。
[Example 2]
<Identification of Pseudomonas sp. MYK105 (Accession No. NITE P-02061) strain and Pseudomonas sp. MYK104 (NITE P-02060) strain>
1) Method
The Pseudomonas sp. MYK105 strain was suspended in 500 μl of a 0.85% aqueous NaCl solution, and the supernatant was removed after centrifugation. To the cells, 20 μl of sterilized water, 25 μl of BL buffer (40 mM Tris, 1% Tween 20, 0.5% Nonidet P-40, 1 mM EDTA, pH 8.0) and 5 μl of Proteinase K (1 mg / ml) were added and lightly suspended. After leaving at 60 ° C. for 20 minutes, it was left at 105 ° C. for 5 minutes. After centrifugation, the supernatant was used as a DNA extract.
滅菌水を6.95μl、Ex Taq Buffer（20ｍM）を1μl、dNTP Mixture（dATP、dCTP、dGTP、dTTP各2.5mM）を0.8μl、Ex Taq(5units/μl)を0.05μl、100nMプライマー（5'-AGAGTTTGATCCTGGCTCAG-3'（配列番号2）及び5'-GGCTACCTTGTTACGACTT-3'（配列番号3））を1μlずつ、滅菌水で10倍に希釈したDNA抽出液１μlを混合し、PCRを行った。反応は94℃、5分間を1サイクル、94℃、60秒間、55℃、60秒間、72℃、90秒間を35サイクル、72℃、2分間を1サイクルで実施した。 6.95 μl of sterile water, 1 μl of Ex Taq Buffer (20 mM), 0.8 μl of dNTP Mixture (2.5 mM for each of dATP, dCTP, dGTP, and dTTP), 0.05 μl of Ex Taq (5 units / μl), and 100 nM primer (5′- 1 μl each of AGAGTTTGATCCTGGCTCAG-3 ′ (SEQ ID NO: 2) and 5′-GGCTACCTTGTTACGACTT-3 ′ (SEQ ID NO: 3) was mixed with 1 μl of a DNA extract diluted 10-fold with sterile water, and PCR was performed. The reaction was carried out in one cycle of 94 ° C. for 5 minutes, 35 cycles of 94 ° C., 60 seconds, 55 ° C., 60 seconds, 72 ° C., 90 seconds, and one cycle of 72 ° C., 2 minutes.
PCR後の溶液をアガロースゲルで電気泳動し、目的のDNA断片部分 (約1400bp)を切り出した。ゲルからDNA断片を抽出し、塩基配列の決定に使用した。決定された塩基配列は、配列番号1の配列からなるものであった。 The solution after PCR was electrophoresed on an agarose gel to cut out a target DNA fragment (about 1400 bp). DNA fragments were extracted from the gel and used for nucleotide sequence determination. The determined base sequence consisted of the sequence of SEQ ID NO: 1.
実施例1で得られたNITE P-02060株についても、上記と同様に16S rDNAの配列を決定した。決定された塩基配列は、配列番号4の配列からなるものであった。 For the NITE P-02060 strain obtained in Example 1, the sequence of 16S rDNA was determined in the same manner as described above. The determined base sequence consisted of the sequence of SEQ ID NO: 4.
２）結果
Pseudomonas sp. MYK105株（NITE P-02061）について、得られた16SrDNAの塩基配列をDDBJ、EMBL、GenBankデータベースと比較した。その結果、相同性の高い菌として、相同性が高いほうから順に、Pseudomonas sp. WXGSA1（Accession No.：KJ184866.1、相同性： 100％）、Pseudomonas koreensis strain JH18（Accession No.：KF424274.1、相同性： 99.9％）、Pseudomonas sp. WXGSY2（Accession No.：KJ184891.1、相同性： 99.9％）、Pseudomonas koreensis strain JH14（Accession No.：KF424272.1、相同性： 99.8％）、Pseudomonas koreensis strain RK18 (Accession No.：KC790278.1、相同性： 99.8％）が認められた。以上から、本細菌はPseudomonas sp. と同定された。また、この結果から、NITE P-02061株は、Pseudomonas koreensisである可能性が高いと考えられた。
2) Results
For Pseudomonas sp. MYK105 strain (NITE P-02061), the nucleotide sequence of the obtained 16S rDNA was compared with DDBJ, EMBL, and GenBank databases. As a result, as bacteria having high homology, Pseudomonas sp. , Homology: 99.9%), Pseudomonas sp. WXGSY2 (Accession No .: KJ184891.1, homology: 99.9%), Pseudomonas koreensis strain JH14 (Accession No .: KF424272.1, homology: 99.8%), Pseudomonas koreensis strain RK18 (Accession No .: KC790278.1, homology: 99.8%) was observed. Based on the above, the bacterium was identified as Pseudomonas sp. From the results, NITE P-02061 strain was considered to be highly likely to be Pseudomonas koreensis.
同様に、Pseudomonas sp. MYK104株（NITE P-02060）についても、得られた16S rDNAの塩基配列をDDBJ、EMBL、GenBankデータベースと比較した。その結果、相同性の高い菌として、Pseudomonas koreensis strain NHZ12 (Accession No.: KJ875595.1、相同性： 100%)、Pseudomonas koreensis strain SMs14 (Accession No.: JX485811.1、相同性： 100%)、Pseudomonas koreensis strain PSB24 (Accession No.: KJ875683.1、相同性： 100%)、Pseudomonas koreensis strain IARI-HHS2-32 (Accession No.: KF054782.1、相同性： 100%)、Pseudomonas sp. BF1-3 (Accession No.: KJ849233.1、相同性： 100%)が認められた。以上から、本細菌はPseudomonas sp. と同定された。また、この結果から、NITE P-02060株は、Pseudomonas koreensisである可能性が高いと考えられた。 Similarly, for Pseudomonas sp. Strain MYK104 (NITE P-02060), the nucleotide sequence of the obtained 16S rDNA was compared with the DDBJ, EMBL, and GenBank databases. As a result, as bacteria having high homology, Pseudomonas koreensis strain NHZ12 (Accession No .: KJ875595.1, homology: 100%), Pseudomonas koreensis strain SMs14 (Accession No .: JX485811.1, homology: 100%), Pseudomonas koreensis strain PSB24 (Accession No .: KJ875683.1, homology: 100%), Pseudomonas koreensis strain IARI-HHS2-32 (Accession No .: KF054782.1, homology: 100%), Pseudomonas sp. BF1-3 (Accession No .: KJ849233.1, homology: 100%). Based on the above, the bacterium was identified as Pseudomonas sp. From the results, it was considered that NITE P-02060 strain was highly likely to be Pseudomonas koreensis.
［実施例３］
＜NITE P-02061株によるタマネギの生育促進１＞
１）方法
タマネギ品種：北もみじ2000
エンドファイト菌株：NITE P-02061株
接種日：平成22年4月27日
接種方法：1×108個／mlの菌懸濁液を500ml/育苗箱（3箱／処理区）に潅注処理した。
定植日：平成22年5月6日
圃場：北海道空知地区
測定項目：収穫期に2条を5mの範囲（3.0m2）で3箇所全量収穫し各規格の個数および重量を調査した。
収穫調査日：平成22年9月4日
[Example 3]
<Onion growth promotion 1 by NITE P-02061 strain>
1) Method Onion varieties: Kitami Momiji 2000
Endophyte strain: NITE P-02061 strain Inoculation date: April 27, 2010 Inoculation method: 1 × 10 8 cell / ml bacterial suspension was irrigated into 500 ml / seedling raising box (3 boxes / treatment area) .
Settlement date: May 6, 2010 Field: Sorachi area, Hokkaido Measurement items: In the harvest season, all 2 strips were harvested at 5 locations within a range of 5 m (3.0 m 2 ), and the number and weight of each standard were investigated.
Harvest survey date: September 4, 2010
２）結果
全量収穫し、各規格の個数と重量の測定を行なった（表1）。大きさ、外観から2L、L大、L、Mに分類された球を規格内とし、それ以外の小さいものや外観が悪いものは規格外とした。
2) Results The whole was harvested, and the number and weight of each standard were measured (Table 1). The spheres classified into 2L, large L, L and M according to their size and appearance were within the standard, and other small or poorly-appearing spheres were outside the standard.
収量調査の規格内収量の結果、NITE P-02061処理区においてはL大、L球の割合が無処理区と比較して大きくなり、収量が21.3％増加した（表2）。 As a result of the yield within the specification of the yield survey, the proportion of L-large and L-spheres was larger in the NITE P-02061-treated section than in the non-treated section, and the yield increased by 21.3% (Table 2).
［実施例４］
＜NITE P-02061株によるタマネギの生育促進２＞
１）方法
タマネギ品種：ターボ
エンドファイト菌株：NITE P-02061株
接種日：平成23年11月
接種方法：1×108個／mlの菌懸濁液を圃場にてスプレーで接種した。
定植日：平成23年11月
圃場：愛知県
測定項目：収穫期に2条を2mの範囲（0.8m2）で3箇所全量収穫し各規格の個数および重量を調査した。
収穫調査日：平成24年6月19日
[Example 4]
<Onion growth promotion 2 by NITE P-02061 strain>
1) Method Onion cultivar: Turbo endophyte strain: NITE P-02061 Inoculation date: November 2011 Inoculation method: A bacterial suspension of 1 × 10 8 cells / ml was inoculated by spraying in a field.
Settlement date: November 2011 Field: Aichi Prefecture Measurement items: During the harvesting season, two lines were harvested at three locations within a range of 2 m (0.8 m 2 ), and the number and weight of each standard were investigated.
Harvest survey date: June 19, 2012
２）結果
全量収穫し、各規格の個数と重量の測定を行なった（表3）。大きさ、外観から2L、L、M、Sに分類された球を規格内とし、それ以外の小さいものや外観が悪いものは規格外とした。
2) Result The whole amount was harvested, and the number and weight of each standard were measured (Table 3). The spheres classified into 2L, L, M, and S according to their size and appearance were within the standard, and the other small or poorly-appearing spheres were outside the standard.
収量調査の規格内収量の結果、NITE P-02061処理区においてはL、M球の割合が無処理区と比較して大きくなり、収量が11.1％増加した（表4）。 As a result of the yield within the specification of the yield survey, the ratio of L and M spheres was larger in the NITE P-02061-treated group than in the untreated group, and the yield was increased by 11.1% (Table 4).
［実施例５］
＜NITE P-02061株によるタマネギの生育促進３＞
１）方法
タマネギ品種：ターボ
エンドファイト菌株：NITE P-02061株
接種日：平成24年11月20日
接種方法：定植前日の苗の根部を1×108個／mlの菌懸濁液一晩漬けた。
定植日：平成24年11月21日
圃場：愛知県
測定項目：収穫期に2条を2mの範囲（0.8m2）で3箇所全量収穫し各規格の個数および重量を調査した。
収穫調査日：平成25年6月18日
[Example 5]
<Onion growth promotion 3 by NITE P-02061 strain>
1) Method Onion varieties: Turbo end fight strain: NITE P-02061 Inoculation date: November 20, 2012 Inoculation method: 1 × 10 8 seeds / ml of bacterial suspension overnight before seedling before planting Pickled.
Planting date: November 21, 2012 Field: Aichi Prefecture Measurement items: All the two lines were harvested at 2 locations (0.8 m 2 ) in the harvest season, and the number and weight of each standard were investigated.
Harvest survey date: June 18, 2013
２）結果
全量収穫し、各規格の個数と重量の測定を行なった（表5）。大きさ、外観から2L、L、M、Sに分類された球を規格内とし、それ以外の小さいものや外観が悪いものは規格外とした。
2) Results The whole quantity was harvested, and the number and weight of each standard were measured (Table 5). The spheres classified into 2L, L, M, and S according to their size and appearance were within the standard, and the other small or poorly-appearing spheres were outside the standard.
収量調査の規格内収量の結果、NITE P-02061処理区においては2L、L球の割合が無処理区と比較して大きくなり、収量が15.3％増加した（表6）。 As a result of the yield within the specification of the yield survey, the ratio of 2L and L spheres was larger in the NITE P-02061-treated group than in the untreated group, and the yield increased by 15.3% (Table 6).
［実施例６］
＜NITE P-02061株によるタマネギの生育促進４＞
１）方法
タマネギ品種：ターボ
エンドファイト菌株：NITE P-02061株
接種日：平成25年10月26日
接種方法： 1×108個／mlの菌懸濁液を苗に潅注処理した。
定植日：平成25年11月20日
圃場：愛知県
測定項目：収穫期に2条を2mの範囲（0.8m2）で3箇所全量収穫し各規格の個数および重量を調査した。
収穫調査日：平成26年6月6日
[Example 6]
<Onion growth promotion 4 by NITE P-02061 strain>
1) Method Onion varieties: Turbo endophyte strain: NITE P-02061 strain Inoculation date: October 26, 2013 Inoculation method: 1 × 10 8 bacterial suspension was irrigated into seedlings.
Planting date: November 20, 2013 Field: Aichi Prefecture Measurement items: Two harvests were harvested at three locations within a range of 2 m (0.8 m 2 ) during the harvest season, and the number and weight of each standard were investigated.
Harvest survey date: June 6, 2014
２）結果
全量収穫し、各規格の個数と重量の測定を行なった（表7）。大きさ、外観から2L、L、M、Sに分類された球を規格内とし、それ以外の小さいものや外観が悪いものは規格外とした。
2) Results The whole was harvested, and the number and weight of each standard were measured (Table 7). The spheres classified into 2L, L, M, and S according to their size and appearance were within the standard, and the other small or poorly-appearing spheres were outside the standard.
収量調査の規格内収量の結果、NITE P-02061処理区においては2L、L球の割合が無処理区と比較して大きくなり、収量が8.3％増加した（表8）。 As a result of the yield within the specification of the yield survey, the ratio of 2L and L-spheres was larger in the NITE P-02061-treated group than in the non-treated group, and the yield was increased by 8.3% (Table 8).
［実施例７］
＜NITE P-02061株によるタマネギの軟腐病の防除＞
１）方法
タマネギ品種：北もみじ2000
エンドファイト菌株：NITE P-02061株
接種日：平成22年4月27日
接種方法：1×108個／mlの菌懸濁液を500ml/育苗箱（3箱/処理区）に潅注処理した。
定植日：平成22年5月6日
圃場：北海道空知地区
測定項目：収穫期に2条を5mの範囲（3.0m2）で3箇所全量収穫し各規格の個数および重量を調査した。その際、乾腐病の病徴が見られる球数を目視により計測した。
収穫調査日：平成22年9月4日
[Example 7]
<Control of soft rot of onion by NITE P-02061 strain>
1) Method Onion varieties: Kitami Momiji 2000
Endophyte strain: NITE P-02061 strain Inoculation date: April 27, 2010 Inoculation method: 1 × 10 8 cells / ml bacterial suspension was irrigated into 500 ml / seedling raising boxes (3 boxes / treatment area) .
Settlement date: May 6, 2010 Field: Sorachi area, Hokkaido Measurement items: In the harvest season, all 2 strips were harvested at 5 locations within a range of 5 m (3.0 m 2 ), and the number and weight of each standard were investigated. At that time, the number of spheres showing signs of dry rot was visually measured.
Harvest survey date: September 4, 2010
２）結果
病害調査を行なった結果を表9に示した。また、収穫された全球数中の被害率を算出し、無処理区を100％としたときのNITE P-02061区の百分率を算出した。その結果、無処理区の被害率を100％とするとNITE P-02061区の被害率は53.1％であり、NITE P-02061処理が、被害を軽減していることが示唆された。
2) Results Table 9 shows the results of the disease survey. In addition, the damage rate in the total number of harvested spheres was calculated, and the percentage of the NITE P-02061 plot was calculated when the untreated plot was taken as 100%. As a result, assuming that the damage rate in the untreated area was 100%, the damage rate in the NITE P-02061 area was 53.1%, suggesting that the NITE P-02061 treatment reduced the damage.
［実施例８］
＜NITE P-02060株による圃場におけるキャベツの生育促進１＞
１）方法
キャベツ品種：親藍及びわかしお
エンドファイト菌株：NITE P-02060株
接種日：平成25年8月10日（親藍）、平成25年9月1日（わかしお）
定植日：平成25年8月14-15日（親藍）、平成25年9月5日（わかしお）
収穫日：平成25年11月8日（親藍）、平成25年12月12日（わかしお）
接種方法：1×108個/mlの菌懸濁液を500ml／育苗箱潅注処理
試験圃場：静岡県磐田市
試験規模：育苗箱4箱(512株)
測定項目：球径（結球しているキャベツの最大直径の平均値）、全収量（地際部で刈取った際の、結球していない葉も含めた全重量の平均値）、及び調整重量（結球していない葉を取り除き、出荷する状態の重量の平均値）
Example 8
<Promotion of cabbage growth in the field by NITE P-02060 strain 1>
1) Method Cabbage varieties: Shinai and Wakashio endophyte strains: NITE P-02060 strain Inoculation date: August 10, 2013 (Shinai), September 1, 2013 (Wakashio)
Settlement date: August 14-15, 2013 (Shinai), September 5, 2013 (Wakashio)
Harvest date: November 8, 2013 (Shinai), December 12, 2013 (Wakashio)
Inoculation method: 1 × 10 8 cells / ml of bacterial suspension in 500 ml / seedling box irrigation test Field: Iwata City, Shizuoka Prefecture Scale: 4 seedling boxes (512 strains)
Measurement items: ball diameter (average value of the maximum diameter of head cabbage), total yield (average of the total weight including non-headed leaves when cut at the ground), and adjusted weight (Average value of the weight in the state when removing non-headed leaves and shipping)
２）結果
各処理区24株を測定した結果を以下の表10及び11に示した。括弧内の数値は、無接種区を100としたときの相対値である。NITE P-02060接種区で球径が大きくなっており、生育が促進されたことが示された。また全重量及び調整重量において1球重量が増加しており、収量も増加したことが示された。
2) Results The results of measuring 24 strains in each treatment group are shown in Tables 10 and 11 below. The numerical values in parentheses are relative values when the uninoculated group was set to 100. In the NITE P-02060 inoculated plot, the diameter of the sphere was larger, indicating that growth was promoted. In addition, the total weight and the adjusted weight increased the weight of one bulb, indicating that the yield also increased.
［実施例９］
＜NITE P-02060株による圃場におけるキャベツの生育促進２＞
１）方法
キャベツ品種：おきな
エンドファイト菌株：NITE P-02060株
接種日：平成26年6月3日
定植日：平成26年6月8日
収穫日：平成26年8月25日
接種方法：1×108個/mlの菌懸濁液を500ml／育苗箱潅注処理
試験圃場：北海道鹿追町
試験規模：育苗箱1箱(128株)
測定項目：実施例８と同様に測定を行った。
[Example 9]
<Promotion of cabbage growth in the field by NITE P-02060 strain 2>
1) Method Cabbage varieties: Okina endophyte strains: NITE P-02060 strain Inoculation date: June 3, 2014 Planting date: June 8, 2014 Harvest date: August 25, 2014 Inoculation method: 1 × 10 8 cells / ml bacterial suspension 500ml / seedling box irrigation test Field: Shikaoi town, Hokkaido Scale: 1 seedling box (128 strains)
Measurement item: Measurement was performed in the same manner as in Example 8.
２）結果
各処理区24株を測定した結果を以下の表12に示す。括弧内の数値は、無接種区を100としたときの相対数である。実施例8と同様にNITE P-02060接種区で球径が大きくなっており、生育が促進されたことが示された。また、全重量及び調整重量において1球重量が増加しており、収量も増加したことが示された。
2) Results The results of measuring 24 strains in each treatment section are shown in Table 12 below. Numerical values in parentheses are relative numbers when the non-inoculated group is 100. As in Example 8, the diameter of the sphere was increased in the NITE P-02060-inoculated plot, indicating that growth was promoted. In addition, it was shown that the weight of one ball increased in the total weight and the adjusted weight, and the yield also increased.
［実施例１０］
＜NITE P-02060株による圃場におけるキャベツの虫害抵抗性の向上＞
１）方法
実施例9において圃場で栽培したキャベツ（おきな）について、平成26年7月18日（定植後40日目）の時点で、各処理区90株程度について、目視により株ごとの食害痕の有無でヨトウムシによる食害を評価した。
[Example 10]
<Improvement of insect resistance of cabbage in the field by NITE P-02060 strain>
1) Method About cabbage (Okina) cultivated in the field in Example 9, as of July 18, 2014 (40th day after planting), about 90 plants in each treatment area were visually inspected for damage to each plant. The damage to the caterpillar was evaluated by the presence or absence of scars.
２）結果
定植後40日目のキャベツを調査した結果、ヨトウムシによる食害株数、食害株率はNITE P-02060接種区で約56％減少した（表13）。
2) Results As a result of investigating cabbage on the 40th day after planting, the number and rate of caterpillars damaged by armyworm decreased by about 56% in the NITE P-02060 inoculated plot (Table 13).
［実施例１１］
＜NITE P-02060株による圃場におけるキャベツの病害抵抗性の向上＞
１）方法
実施例8において圃場で栽培したキャベツ（親藍）について、収穫日（平成25年11月8日）におけるアブラナ科根こぶ病の罹病程度を測定した。具体的には、各処理区100株について、目視により株ごとの根こぶの罹病程度を5 段階に評価し(0:罹病率0%、1:罹病率1〜25%、2:罹病率26〜50%、3:罹病率51〜75%、4:罹病率76〜100%)、評価した値を用いて下記の計算式で根こぶ指数を算出して評価した。
根こぶ指数＝(4A＋3B＋2C＋D)/4×100
[A:4の株数,B:3の株数,C:2の株数,D:1の株数]
[Example 11]
<Improvement of disease resistance of cabbage in the field by NITE P-02060 strain>
1) Method With respect to cabbage (parent indigo) cultivated in the field in Example 8, the degree of scab attack on the harvest date (November 8, 2013) was measured. Specifically, for 100 treatments in each treatment area, the degree of root knot disease was visually evaluated for each strain in five stages (0: morbidity 0%, 1: morbidity 1 to 25%, 2: morbidity 26 -50%, 3: morbidity rate 51-75%, 4: morbidity rate 76-100%), and using the evaluated values, a root-knot index was calculated by the following formula and evaluated.
Root-knot index = (4A + 3B + 2C + D) / 4 x 100
[A: 4 shares, B: 3 shares, C: 2 shares, D: 1 shares]
２）結果
無接種区を100としたときの被害度がNITE P-02060株接種区で12.3%減少した（表14）。
2) Results The degree of damage was reduced by 12.3% in the NITE P-02060 strain inoculated group when the uninoculated group was set to 100 (Table 14).
［実施例１２］
＜NITE P-02060株によるポット試験におけるコマツナの生育促進＞
１）方法
コマツナ品種：夏楽天
エンドファイト菌株：NITE P-02060株
試験規模：1処理区10株、3反復
5×5セル（計25セル）になるように準備したセルトレーの底に約2cm角に切った不織布を引き、タネマキ培土（タキイ種苗株式会社）を充填した。2粒／セルずつ播種し、タネマキ培土で覆土した。播種日を0日目として播種後7日目前後に間引きをして1株／セルにした。NITE P-02060株接種区には、播種後14日を目安に本葉が2〜3枚展開したところで、1×108cells/mlに純水を用いて調整した菌液を1ml／セルずつ接種した。15℃以下にならないように加温したガラス温室内で栽培し本葉が4枚以上展開するまで栽培した。その後地際部から切断し、双葉を取り除いた後、乾物重を測定した。
[Example 12]
<Promotion of Komatsuna growth in pot test by NITE P-02060 strain>
1) Method Komatsuna cultivar: Natsu Rakuten end fight strain: NITE P-02060 strain Test scale: 10 strains per treatment plot, 3 repetitions
A nonwoven fabric cut into about 2 cm squares was drawn on the bottom of a cell tray prepared so as to have 5 × 5 cells (total 25 cells), and filled with seed cultivation soil (Takii Seed Co., Ltd.). Two seeds per cell were seeded and covered with seed cultivation. With the seeding date as day 0, thinning was performed around day 7 after seeding to obtain 1 strain / cell. In the inoculated plot of NITE P-02060 strain, when 2 to 3 true leaves were developed on the 14th day after seeding, 1 × 10 8 cells / ml of bacterial solution adjusted using pure water at 1 ml / cell Inoculated. Cultivation was carried out in a glass greenhouse heated to not more than 15 ° C. until four or more true leaves were developed. Thereafter, cutting was performed from the ground part, and after removing the futaba, the dry weight was measured.
２）結果
各処理区3回反復試験を行った結果、無接種区を100としたときにNITE P-02060接種区で乾物重が8.8%増加していた（表15）。
2) Results As a result of repeating the test three times in each treatment group, the dry matter increased by 8.8% in the NITE P-02060 inoculation group when the non-inoculation group was set to 100 (Table 15).
［実施例１３］
＜NITE P-02060株によるポット試験におけるレタスの生育促進＞
１）方法
レタス（品種：グリーンウェーブ）を用いる以外は、実施例１２と同様に試験を行い、乾物重を測定した。
Example 13
<Promotion of lettuce growth in pot test by NITE P-02060 strain>
1) Method A test was performed in the same manner as in Example 12 except that lettuce (variety: green wave) was used, and the dry weight was measured.
２）結果
各処理区3回の反復試験を行った結果、無接種区を100としたときにNITE P-02060株接種区で乾物重が66.6%増加したことが示された（表16）。
2) Results As a result of repeating the test three times in each treatment group, it was shown that the dry matter increased by 66.6% in the NITE P-02060 strain inoculation group when the non-inoculation group was set to 100 (Table 16).
［実施例１４］
＜NITE P-02060株によるポット試験におけるタマネギの生育促進＞
１）方法
タマネギ（品種：ネオアース）を用いる以外は、実施例１２と同様に試験を行い、乾物重を測定した。
[Example 14]
<Promotion of onion growth in pot test by NITE P-02060 strain>
1) Method A test was performed in the same manner as in Example 12 except that onion (variety: Neoearth) was used, and the dry weight was measured.
２）結果
各処理区3回反復試験を行った結果、無接種区を100としたときにNITE P-02060株接種区で乾物重が4.5%増加していた（表17）。
2) Results As a result of three repetitions of each treatment group, the dry matter increased by 4.5% in the NITE P-02060 strain inoculation group when the non-inoculation group was set to 100 (Table 17).
［実施例１５］
＜NITE P-02060株によるポット試験におけるトマトの生育促進＞
１）方法
トマト（品種：CF桃太郎ヨーク）を用いる以外は、実施例１２と同様に試験を行い、乾物重を測定した。
[Example 15]
<NITE P-02060 strain promotes tomato growth in pot test>
1) Method Except for using tomato (variety: CF Momotaro York), a test was performed in the same manner as in Example 12, and the dry weight was measured.
２）結果
各処理区3回の反復試験を行った結果、無接種区を100としたときにNITE P-02060株接種区で乾物重が3.2%増加した（表18）。
2) Results As a result of repeating the test three times in each treatment group, the dry matter increased by 3.2% in the NITE P-02060 strain inoculation group when the non-inoculation group was set to 100 (Table 18).
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